Glycoglycerophospholipid, antibody thereagainst, and method for detecting mycoplasma

ABSTRACT

Mouse is immunized with an antigen of a lipid fraction originating from  Mycoplasma fermentans . Its spleen cells are fused with mouse myeloma cells to prepare hybridomas. A hybridoma is selected, which produces a monoclonal antibody having reaction specificity to GGPL-III that is a phosphocholine-containing glycoglycerolipid specific to  Mycoplasma fermentans. Mycoplasma fermentans  is detected by using the obtained antibody.

This application is a divisional of prior application Ser. No.08/750,677, filed Dec. 3, 1996 now U.S. Pat. No. 5,994,090.

TECHNICAL FIELD

The present invention relates to a novel glycoglycerophospholipidoriginating from Mycoplasma fermentans, an antibody against theglycoglycerophospholipid specifically existing in Mycoplasma fermentans,a method for measuring the glycoglycerophospholipid based on the use ofthe antibody, and a method for detecting Mycoplasma fermentans.

BACKGROUND ART

The present inventors have already found five species ofglycoglycerophospholipids (phosphocholine-containing glycoglycerolipids)from MT-4 cells (human helper T cells infected with HTLV-I (human Tlymphotropic retrovirus Type I), Miyoshi et al., Gann., 71, 155-156(1980)). The present inventors have previously found that one of thefive species of glycoglycerophospholipids is6′-O-phosphocholine-α-glucopyranosyl-(1′-3)-1,2-diacyl-sn-glycerol(Shishitsu-Seikagaku-Kenkyu (Studies on Lipid Biochemistry), Vol. 35,pp. 111-114, 1993).

On the other hand, it has been reported that Macodlasma fermentans is anexacerbation factor of human acquired immunodeficiency syndrome (AIDS)(Lo. S., -C. et al., 1991, Science, 251: 1074-1076; U.S. Pat. No.5,242,820), or Mycoplasma fermentans is a cause of rheumatism (Williams,M. H. et al., 1970, Lancet ii: 277-280).

Various antibodies against mycoplasmas have been hitherto known, andthey have been also used for clinical examination. However, the majorityof them are antibodies against Mycoplasma pneumoniae or Mycoplasmagenitalium. Monoclonal antibodies against these mycoplasmas have beenalso prepared. However, it is presumed that such an antibody is anantibody which recognizes a protein of a mycoplasma, or simultaneouslyrecognizes a protein and a lipid of a mycoplasma. Further, any of suchantibodies does not exhibit specificity to Mycoplasma fermentans(Japanese Patent Laid-open Nos. 63-298, 63-184064, 63-32496, and5-304990, and U.S. Pat. Nos. 5,158,870, 4,945,041, and 5,242,820). Anantibody, which exhibits specificity to Mycoplasma fementans, isdisclosed in U.S. Pat. No. 5,242,820. However, this antibody is obtainedby using an entire extract of mycoplasmal cells as an immunogen, andthus the antibody is regarded as an antibody which recognizes a protein.Accordingly, if a mycoplasma contained in a body fluid such as a serumwhich contains various proteins is detected by using this antibody, theantibody highly possibly makes nonspecific binding. Therefore, it may beimpossible to expect a high sensitivity. Further, when an antigen is aprotein, it is sufficiently assumed that antigenicity disappears due tomutation in an amino acid sequence of the protein.

As far as the present inventors know, it has not been reported that anymycoplasma has a glycoglycerophospholipid containing phosphocholine.Further, no instance has been known, in which a glycoglycerophospholipidoriginating from a mycoplasma is used as an immunogen to obtain anantibody which exhibits specificity to the glycoglycerophospholipid.Moreover, it has not been known at all as well that the antibody, whichexhibits the specificity to the glycoglycerophospholipid, exhibits highspecificity to Mycoplasma fementans.

DISCLOSURE OF THE INVENTION

It has been reported that the process of pathology of a patient infectedwith a human immunodeficiency virus is accelerated to arrive at AIDS byinfection of a mycoplasma (1991, Science, 251: 4991). It has been alsoreported that Mycoplasma fementans is an exacerbation factor of AIDS asdescribed above. However, there is no means to correctly detect suchbehavior of Mycoplasma fermentans in vivo. Therefore, it has beendesired to provide an antibody capable of immunologically detectingMycoplasma fementans in vivo.

The present invention has been made considering a viewpoint as describedabove, an object of which is to elucidate a glycoglycerophospholipidspecifically existing in Mycoplasma fementans, and provide an antibodyagainst the glycoglycerophospholipid, a method for measuring theglycoglycerophospholipid based on the use of the antibody, and a methodfor detecting Mycoplasma fementans.

In order to elucidate abnormal proliferation of cells, destruction ofcells, and immunological abnormality caused by infection with aretrovirus, the present inventors have analyzed lipids of such infectedcells. During this process, the present inventors have confirmed that aglycoglycerophospholipid (phosphocholine-containing glycoglycerolipid)extracted from a human helper T cell strain is unexpectedly aglycoglycerophospholipid originating from Mycoplasma fermentans(phosphocholine-containing glycoglycerolipid: hereinafter simplyreferred to as “glycoglycerophospholipid). Further, the presentinventors have elucidated a structure of the lipid. Moreover, thepresent inventors have prepared an antibody against the lipid, andconfirmed specificity to various mycoplasmas. As a result, the presentinventors have found out that the antibody specifically recognizesMycoplasma fementans. Thus the present invention has been completed.

Namely, the present invention lies in a glycoglycerophospholipidextractable from Mycoplasma fermentans, having the following properties:

(A) the glycoglycerophospholipid is reactive with orcinol reagent,Dittmer reagent, Dragendorff reagent, and ninhydrin reagent;

(B) the glycoglycerophospholipid is degradable with alkali;

(C) the glycoglycerophospholipid is obtained as a non-adsorptivefraction upon fractionation with an anion exchanger havingdiethylaminoethyl group; and

(D) the glycoglycerophospholipid has a molecular weight of 1048+28nmeasured by using a mass spectrometer, wherein n is −1, 0, 1, or 2.

The glycoglycerophospholipid described above highly possibly comprisesconstitutional components ofα-glucopyranosyl-(1′-3)-1,2-diacyl-sn-glycerol, phosphocholine, andphosphoric ester of aminopropanediol.

In another aspect, the present invention lies in ananti-glycoglycerophospholipid antibody having reaction specificity to aglycoglycerophospholipid comprising at least phosphocholine, glucose,fatty acid, and glycerol, the lipid being non-adsorptive to an anionexchanger having diethylaminoethyl group, and unstable against alkali.The glycoglycerophospholipid includes, for example, aglycoglycerophospholipid specifically existing in Mycoplasma fementans.

The present invention provides, as specified embodiments of theanti-glycoglycerophospholipid antibody, an anti-glycoglycerophospholipidpolyclonal antibody which reacts with both of 6′-O-phosphocholine-α-glucopyranosyl-(1′-3)-1,2-diacyl-sn-glycerol and theglycoglycerophospholipid extractable from Mycoplasma fermentans, havingthe following properties, and an anti-glycoglycerophospholipidmonoclonal antibody which has reaction specificity to theglycoglycerophospholipid having the following properties:

(A) the glycoglycerophospholipid is reactive with orcinol reagent,Dittmer reagent, Dragendorff reagent, and ninhydrin reagent;

(B) the glycoglycerophospholipid is degradable with alkali;

(C) the glycoglycerophospholipid is obtained as a non-adsorptivefraction upon fractionation with an anion exchanger havingdiethylaminoethyl group; and

(D) the glycoglycerophospholipid has a molecular weight of 1048+28nmeasured by using a mass spectrometer, wherein n is −1, 0, 1, or 2.

In further aspects, the present invention provides a method formeasuring a glycoglycerophospholipid, comprising the step ofimmunologically measuring the glycoglycerophospholipid having theforegoing properties contained in a specimen, by using the foregoinganti-glycoglycerophospholipid antibody, and a method for detectingMycoplasma fementans, comprising the steps of measuring aglycoglycerophospholipid contained in a specimen in accordance with theforegoing method, and relating the presence or absence of theglycoglycerophospholipid or an existing amount thereof to the presenceor absence of Mycoplasma fementans or an existing amount thereof in thespecimen.

In still another aspect, the present invention provides a method formeasuring the glycoglycerophospholipid having the foregoing properties(A) to (D) and/or a substance having antigenicity similar to that of theglycoglycerophospholipid contained in a specimen, comprising the step ofimmunologically measuring the glycoglycerophospholipid and/or thesubstance having the antigenicity similar to that of theglycoglycerophospholipid by using the foregoinganti-glycoglycerophospholipid antibody.

In still another aspect, the present invention provides a reagent kitfor detecting Myconlasma fermentans or a glycoglycerophospholipid ofMycoplasma fermentans contained in a specimen in accordance with animmunological method, comprising the foregoinganti-glycoglycerophospholipid antibody, and a glycoglycerophospholipidof Mycoplasma fementans labeled with a label substance, or a secondaryantibody obtained by labeling an antibody against immunoglobulin of animmunized animal with a label substance, the antibody against theimmunoglobulin of the immunized animal being prepared by using an animalother than the immunized animal used to prepare theanti-glycoglycerophospholipid antibody.

In still another aspect, the present invention provides a method fordetecting a disease selected from AIDS, nephritis, and HTLV-I associatedmyelopathy, comprising the step of detecting theglycoglycerophospholipid having the foregoing properties (A) to (D), asubstance having antigenicity similar to that of theglycoglycerophospholipid, or an antibody having reaction specificity tothe glycoglycerophospholipid contained in blood.

In this specification, the glycoglycerophospholipid specificallyexisting in Mycoplasma fementans is simply referred to as“glycoglycerophospholipid”, if necessary.

The present invention will be explained in detail below.

<1> Glycoglycerophospholipid of the present invention

The antibody of the present invention has reaction specificity to aglycoglycerophospholipid of Mycoplasma fermentans, which is prepared byusing the glycoglycerophospholipid as an antigen. At first, theglycoglycerophospholipid of Mycoplasma fementans will be explained.

Existence of lipids inherent in cells infected with a retrovirus hasbeen hitherto known. Five species of glycoglycerophospholipids (GGPLs:GGPL-I, GGPL-II, GGPL -III, GGPL-IV, GGPL-V) have been found in humanhelper T cells infected with HTLV-I. Among them, the structure of GGPL-Ihas been determined by the present inventors (Nishida et al., NipponNogeikagaku Kaishi, Vol. 68, No. 3 (1994), “Proceedings of 1994th AnnualMeeting”, p. 39). As for GGPLs other than GGPL-I, only their existenceis acknowledged, and neither their physical properties nor theirstructures have been known.

The present inventors prepared lipid fractions from HTLV-I-infectedhuman helper T cells (MT-4 (GGPL+)) in which GGPLs were found, and fromcells of MT-4 (GGPL−) obtained by treating the cells of MT-4 (GGPL+)with an anti-mycoplasmal agent (MC201, produced by DainipponPharmaceutical). The prepared lipid fractions were separated by HPTLC(high-performance thin layer chromatography). Glycolipid was stainedwith the orcinol reagent, and phospholipid was stained with Dittmerreagent. As a result, two bands were detected (FIG. 4), which were foundin MT-4 (GGPL+), and were not found in MT-4 (GGPL−). The two bands werealso detected from MT-4 (GGPL−) cells obtained by cultivation withaddition of a culture supernatant of MT-4 (GGPL+) cells previouslypassed through a filter having a pore size of 0.22 μm. As explained inExamples described later on, it was found that one of the two bands wasGGPL-I, and the other was GGPL-III.

It has not been reported that any microorganism belonging to the genusMycoplasma has, as a constitutional component, aglyceroglycophospholipid containing phosphocholine. It has beenconsidered that GGPLs originate from human cells. However, it has beenclarified from the result described above that GGPLs originate fromMycoplasma fementans. Therefore, it is assumed that Mycoplasma fementanscan be detected if an antibody having reaction specificity to theglyceroglycophospholipid described above is obtained.

The glycoglycerophospholipid of Mycoplasma fermentans can be preparedfrom a culture of Mycoplasma fermentans. Mycoplasma fementans can beobtained, for example, as follows. A culture supernatant of culturedcells in which existence of GGPLs is confirmed (“GGPL -positive”), forexample, a culture supernatant of MT-4 cells (human helper T cellsinfected with HTLV-I (human T lymphotropic retrovirus Type I)) isfiltrated with a filter having a pore size of 0.22 μm. Thus Mycoplasmafermentans is obtained in a filtrate. Alternatively, it is alsoallowable to use a type strain such as Mycoplasma fementans PG18 strain.

Obtained Mycoplasma fementans is cultivated with, for example, an agarmedium of PPLO broth (produced by Difco Laboratories) to isolate acolony which is then cultivated in a liquid medium such as PPLO broth(produced by Difco Laboratories) containing 10% (v/v) fetal bovine serum(FBS), 5% (w/v) yeast extract (produced by Flow Laboratories), 1,000units/ml penicillin, 1% (w/v) dextrose, and 0.002% (w/v) phenol red.Thus cultivated microbial cells of Mycoplasma fermentans are obtained.Next, methanol is added to the microbial cells,of Mycoplasma fementans,followed by being left to stand for several hours. The thus-obtainedmicrobial cells are then subjected to ultrasonic treatment afterchloroform is added thereto. The ultrasonic treated microbial cells isallowed to stand for several hours—therefor. Subsequently, the microbialcells are homogenized by using, for example, a Potter type TEFLONhomogenizer to recover a supernatant. A lipid extract is obtained byevaporating the supernatant. The lipid extract thus obtained containsthe glycoglycerophospholipid.

As described in Examples, the lipid fraction was applied to an HPTLC(high-performance thin layer chromatography) plate, which was developedwith a mixed solvent of chloroform: methanol: 0.2% (w/v) calciumchloride aqueous solution=50:45:10 (v/v/v) to analyze phospholipid. As aresult, six bands (Lipid i, Lipid ii, Lipid iii, Lipid iv, Lipid v, andLipid vi) were detected (FIG. 3). According to the behavior on TLC, itwas revealed that Lipid v and Lipid vi of them corresponded to GGPL-Iand GGPL-III respectively. According to a result of FAB massspectrometry, it was confirmed that GGPL-I was identical with Lipid v,and GGPL-III was identical with Lipid vi.

The glycoglycerophospholipid of Mycoplasma fermentans can be alsoobtained from MT-4 cells infected with Mycoplasma fementans. Forexample, MT-4 cells are cultured in RPMI-1640 medium added with 10%(v/v) FCS (fetal calf serum), and obtained cells are washed with PBS(phosphate buffered saline), from which lipids are extracted by using400 ml of a mixed solution of chloroform: methanol (=2:1, 1:1, or 1:2).

The lipid extract of Mycoplasma fementans or MT-4 cells obtained asdescribed above is divided into a non-adsorptive fraction (neutralfraction) and an adsorptive fraction (acid fraction) by using an anionexchange resin having diethylaminoethyl (DEAE) group such asDEAE-Sephadex A25 (produced by Pharmacia), and thus the non-adsorptivefraction is obtained. The non-adsorptive fraction is applied to a silicabead column, and fractionated with a concentration gradient based onchloroform/methanol/water (83:16:0.5 to 20:80:8 (v/v)). Thus GGPL-IIIcan be separated from other phospholipids. Further, GGPL-I is isolatedby performing elution with a concentration gradient based on1-propanol/aqueous ammonia/water (80:5:15 to 75:5:20).

Both of GGPL-I and GGPL-III are positive to the orcinol reagent, Dittmerreagent, and Dragendorff reagent (which stains choline), and they aredegraded by a treatment with mild alkali. GGPL-I is negative to theninhydrin reaction (which stains amino group), however, GGPL-III ispositive to this reaction.

Results of physicochemical analysis performed by using purified GGPL-IIIare shown below.

(1) Infrared Absorption Spectrum

Absorption bands corresponding to —CH₂ and —CH₃ groups, hydroxyl group,estercarbonyl group, phosphate group, choline group, and primary aminegroup were detected respectively.

(2) Liquid Secondary Ionization Mass Spectrometry (LSIMS)

It was suggested that at least three species of fatty acids were presentin the molecule. It was concluded that GGPL-III existed as at least fourspecies of molecules, a major component of which had a molecular weightof 1048. Further, it was demonstrated that species of GGPL-III havingmolecular weights of 1020, 1076, and 1104 were also present.

(3) Tandem Mass Spectrometry (MS/MS)

It was suggested that phosphocholine was present in the molecule. It wasconcluded that the major component of GGPL-III had a molecular weight of1048.

(4) Unidimensional ¹H NMR Spectrum

Signals of glycerol, choline, and glucose were detected. Further, it waspostulated that aminopropanediol was present. Accordingly, the spectrumof GGPL-III was compared with unidimensional ¹H NMR spectrums obtainedby using standard samples of 3-aminopropane-1,2-diol(1-aminopropane-2,3-diol) and 2-aminopropane-1,3-diol. As a result, itwas suggested that GGPL-III possibly contained 2-aminopropane-1,3-diolas a constitutional component.

(5) Two-dimensional ¹H NMR Spectrum (PH-DQF-COSY method)

A signal of proton originating from diacylglycerol was observed. Asignal of proton of choline was also observed.

(6) Two-dimensional ¹H- ³¹P NMR Spectrum

It was demonstrated that one molecule of GGPL-III contained twophosphorus atoms (P). It was postulated that a compound containingphosphorus bound to 1- or 6-position of a glucose residue. This positionwas highly possibly 6-position.

A structure of phosphocholine was suggested, in which a phosphate groupbound to choline. A structure was postulated, in which a phosphate esterof aminopropanediol firstly bound to the 6-position of the glucoseresidue, and phosphocholine bound to the phosphoric ester ofaminopropanediol.

According to the results described above, it has been revealed thatGGPL-III is a novel glycoglycerophospholipid having the followingproperties:

(A) the glycoglycerophospholipid is reactive with orcinol reagent,Dittmer reagent, Dragendorff reagent, and ninhydrin reagent;

(B) the glycoglycerophospholipid is degradable with alkali;

(C) the glycoglycerophospholipid is obtained as a non-adsorptivefraction upon fractionation with an anion exchanger having DEAE group;and

(D) the glycoglycerophospholipid has a molecular weight of 1048+28nmeasured by using a mass spectrometer, wherein n is −1, 0, 1, or 2.

According to the results described above, it was demonstrated thatGGPL-III contained constitutional components ofα-glucopyranosyl-(1′-3)-1,2-diacyl-sn -glycerol, phosphocholine, andphosphate ester of aminopropanediol. The phosphate ester ofaminopropanediol was a phosphate ester of 2-aminopropane-1,3-diol. Itwas postulated that its binding site was 6′-position of the glucosereside of α-glucopyranosyl-(1′-3)-1,2-diacyl-sn-glycerol. It was furthersuggested that phosphocholine bound to the phosphate ester of2-aminopropane-1,3-diol. The major component of GGPL-III had twopalmitoyl groups as acyl groups, having its deduced structure asrepresented by the following formula (I). It was postulated that themajor component of GGPL-III had the structure in which the phosphoricester of 2-aminopropane-1,3-diol was inserted between phosphocholine andthe glucose residue of GGPL-I(6′-O-phosphocholine-α-glucopyranosyl-(1′-3)-1,2-dipalmitoyl-sn-glycerol).

On the other hand, the other GGPL-III molecules having differentmolecular weights involve different types of acyl groups inα-glucopyranosyl-(1′-3)-1,2-diacyl-sn-glycerol. It is postulated thatthe molecule having a molecular weight of 1020 has a myristoyl group anda palmitoyl group, the molecule having a molecular weight of 1076 has apalmitoyl group and a stearoyl group, and the molecule having amolecular weight of 1104 has two stearoyl groups or a palmitoyl groupand an eicosanoyl group. However, details are not clarified.

<2> Preparation of Antibody of the Present Invention

The antibody of the present invention is obtained by immunizing ananimal with an antigen of the glycoglycerophospholipid originating fromMycoplasma fermentans, and separating serum from the animal.Alternatively, the antibody of the present invention is obtained bycollecting antibody-producing cells of the animal, rendering theantibody-producing cells permanently culturable, and recovering theantibody from a culture thereof. The method for preparing the antibodyof the present invention will be exemplarily described below. However,there is no limitation thereto. The antibody of the present inventionmay be prepared in accordance with other methods provided that theglycoglycerophospholipid of Mycoplasma fementans is used as an antigen.

(1) Preparation of Polyclonal Antibody

Monophosphate lipid, Freund's complete adjuvant, and mineral oil areadded to and mixed with the lipid extract of Mycoplasma fementansobtained as described above. PBS (phosphate buffered saline) containing0.1% (v/v) Tween 80 is added thereto and emulsified.

Next, an obtained emulsion is subcutaneously or intraperitoneallyadministered to an animal such as mouse, rat, rabbit, guinea pig, orsheep. After priming immunization, boosting immunization is performedtwo or three weeks later in accordance with an ordinary method. Thus anantiserum having a high titer is obtained. Blood is collected one weekafter the final immunization, and serum is separated. The serum isheat-treated to inactivate complement. After that, an immunoglobulinfraction is obtained in accordance with a method similar to those usedto purify an ordinary antibody, such as salting out with ammoniumsulfate and ion exchange chromatography. Desirably, the increase inantibody titer in blood is confirmed in accordance-with, for example,enzyme immunoassay after the final immunization.

The antibody obtained as described above has reaction specificity to theglycoglycerophospholipid of Mycoplasma fementans, and it does not reactwith glycoglycerophospholipids of other mycoplasmas such as Mycoplasmaarthritidis and Mycoplasma hominis.

A polyclonal antibody having reaction specificity to GGPL-III can beobtained by using purified GGPL-III instead of the lipid extract ofMycoplasma fementans.

(2) Preparation of Monoclonal Antibody

A monoclonal antibody is obtained in accordance with a method of Kohlerand Milstein (Nature, pp. 495-492, 1975). Namely, antibody-producingcells of a mammalian, which produce an antibody against theglycoglycerophospholipid, is fused with myeloma cells to producehybridomas. A hybridoma, which produces an objective antibody, iscloned, and the hybridoma is cultured. Thus the monoclonal antibody isobtained in a culture liquid. This process will be explained below whiledividing the process into respective steps.

(i) Immunization of Animal and Preparation of Antibody-Producing Cells

Cells, which produce the antibody against the glycoglycerophospholipid,are obtained by immunizing an animal such as mouse, rat, rabbit, guineapig, or sheep with the glycoglycerophospholipid, and preparing, forexample, spleen cells, lymph node cells, or peripheral blood from theanimal. The animal may be immunized with the glycoglycerophospholipid inthe same manner as described in the item (1).

The monoclonal antibody having reaction specificity to GGPL-III may beobtained by immunizing an animal with purified GGPL-III. Alternatively,the monoclonal antibody may be obtained by preparing hybridomas by usingantibody-producing cells of an animal immunized with aglycoglycerophospholipid mixture, and selecting a strain from theobtained hybridomas, the strain producing a monoclonal antibody havingreaction specificity to GGPL-III. According to the latter method, it isunnecessary to obtain GGPL-III in an amount required to immunize theanimal. It is sufficient to prepare GGPL-III in a minute amount of adegree capable of performing detection by the aid of enzyme immunoassay.

(ii) Preparation of Hybridoma

Antibody-producing cells are collected from the animal immunized withthe glycoglycerophospholipid to perform cell fusion with myeloma cells.Various mammalian cell strains can be utilized as the myeloma cells tobe used for cell fusion. However, it is preferred to use a cell strainwhich is the same species as the animal used to prepare theantibody-producing cells. In order to distinguish fused cells fromnon-fused cells after cell fusion, it is preferred to use a myeloma cellstrain which has a marker so that non-fused myeloma cells may notsurvive, and only hybridoma cells may proliferate. For example,8-azaguanine-resistant strain is deficient in hypoxanthine guaninephosphoribosyltransferase (HGPRT), and its nucleic acid synthesisdepends on a de novo synthesis pathway. A fused cell (hybridoma) of sucha myeloma cell and a normal antibody-producing cell is proliferative ina medium (HAT medium) containing hypoxanthine, aminopterin, andthymidine because the fused cell can synthesize nucleic acid by using asalvage circuit originating from lymphocyte owing to the presence ofthymidine and hypoxanthine even when the de novo synthesis pathway isinhibited by aminopterin. On the contrary, the myeloma cells resistantto 8-azaguanine cannot synthesize nucleic acid, and the cells diebecause the de novo synthesis pathway is inhibited by aminopterin.Further, the antibody-producing cells as the normal cells cannot becultured for a long period of time. Therefore, only the hybridoma cellsproduced by fusion of the antibody-producing cells and the myeloma cellscan proliferate in the HAT medium. Accordingly, fused cells can beselected from non-fused cells (Science, Vol. 145, p. 709, 1964). Astrain which secretes no inherent immunoglobulin is preferably used asthe myeloma cell, in view of the fact that it is easy to obtain anobjective antibody from a culture supernatant of an obtained hybridoma.

Cell fusion to obtain the hybridoma is performed, for example, asfollows. A spleen is excised from the immunized animal, and it issuspended in RPMI 1640 medium to prepare a cell-floating suspension. Thespleen cells are mixed with mouse myeloma cells such as SP2/0 cells(azaguanine-resistant, IgG-non-secretable: ATCC CRL-1581) at thelogarithmic growth phase so that the ratio of spleen cells to themyeloma cells is about 10:1 to 1:1. After centrifugation, polyethyleneglycol having an average molecular weight of 1,000 to 6,000 is added toa precipitate to give a final concentration of 30 to 50%. Thus thespleen cells and the myeloma cells are fused. Fusion may be performed byapplying electric pulse to the cell mixture instead of the addition ofpolyethylene glycol.

The cells having been subjected to the fusion treatment are culturedwith, for example, RPMI 1640 medium containing 10% (v/v) fetal calfserum (FCS), and then the cells are floated in a selective medium suchas HAT medium. The cells are dispensed and poured into, for example,wells of a 96-well microtiter plate. Thus the cells are cultured so thatonly hybridomas are allowed to grow.

(iii) Screening for Hybridoma which Produces Antibody Having ReactionSpecificity to Glycoglycerophospholipid

The hybridomas obtained as described above are provided as a mixture ofhybridomas which produce monoclonal antibodies against a plurality ofantigens or epitopes respectively. Accordingly, it is significant toselect, from the hybridomas, a strain which produces a monoclonalantibody having reaction specificity to the glycoglycerophospholipid,especially a monoclonal antibody having reaction specificity toGGPL-III. It is preferred to select a strain which produces a monoclonalantibody against an epitope having strong antigenicity, from monoclonalantibodies which bind to GGPL-III.

A strain, which produces a monoclonal antibody against theglycoglycerophospholipid, can be selected in accordance with an enzymeimmunoassay by using the glycoglycerophospholipid as an antigen. Such amethod includes an ELISA method comprising the following steps. Namely,the antigen is immobilized on a solid phase such as a microtiter plate,to which a culture liquid of a hybridoma is added, followed by additionof a secondary antibody labeled with, for example, an enzyme, afluorescent substance, or a luminescent substance to perform incubation. Thus the antibody is detected by the aid of the bound label substance.In this method, an antibody may be immobilized on a solid phase, towhich the antigen and a labeled secondary antibody may be successivelyadded, followed by incubation. The ELISA method will be described indetail later on.

When no purified GGPL-III is obtained, the lipid fraction of Mycoplasmafementans is separated by using a high-performance thin layerchromatography (HPTLC) plate. A culture liquid of a hybridoma and alabeled secondary antibody are successively added to the plate, followedby incubation so that a position at which the label substance makesbinding is detected. If the position is identical with a position ofGGPL-III developed by HPTLC, the hybridoma is regarded to produce amonoclonal antibody against GGPL-III. Once the monoclonal antibodyagainst GGPL-III is obtained, GGPL -III can be also purified from thelipid fraction in accordance with affinity chromatography or the likebased on the use of the monoclonal antibody.

When it is confirmed that a well contains a hybridoma which produces anobjective monoclonal antibody, cloning is performed from cells in thewell containing the hybridoma in accordance with limiting dilutionanalysis or the like.

As demonstrated in Examples described later on, the hybridoma strainthus obtained, which produces the monoclonal antibody having reactionspecificity to GGPL -III, has been deposited on May 24, 1994 in NationalInstitute of Bioscience and Human Technology of Agency of IndustrialScience and Technology of Ministry of International Trade and Industryunder a deposition number of FERM P-14324, transferred to internationaldeposition based on the Budapest Treaty on May 26, 1995, and awarded adeposition number of FERM BP-5115.

(iv) Preparation of Monoclonal Antibody

The monoclonal antibody of the present Invention is obtained in aculture supernatant by culturing the hybridoma obtained as describedabove in an appropriate medium. The monoclonal antibody can be purifiedin accordance with an ordinary method including, for example, saltingout with ammonium sulfate, ion exchange chromatography, affinitychromatography based on the use of protein A or protein G, andimmunoadsorption chromatography based on the use of an immobilizedantigen.

The monoclonal antibody thus obtained makes no cross reaction withsialic acid-containing glycolipid (ganglioside) existing in serum of ahealthy person not infected with Mycoplasma fementans, plateletactivating factor (1-alkyl-2-acetylglycero-3-phosphocholine) or apartially deacylated product thereof, phosphatidylcholine or a partiallydeacylated product thereof, glycolipid, and phospholipid such assphingomyelin.

The monoclonal antibody of the present invention can be used as it is.However, those obtained by fragmentation can be also used. Uponfragmentation of the antibody, it is indispensable for binding betweenthe antigen and the antibody that the antigen-binding site (Fab) of theantibody is conserved. Therefore, it is possible to use a fragmentcontaining the antigen-binding site (Fab), obtained by treating theantibody with a protease (for example, plasmin, pepsin, and papain)which does not degrade the antigen-binding site.

If a nucleotide sequence of a gene coding for the monoclonal antibody ofthe present invention or an amino acid sequence of the antibody isdetermined, it is possible to produce a fragment containing theantigen-binding site (Fab) in accordance with a technique of geneticengineering.

<3> Utilization of Glcoglycerphospholipid and Antibody thereagainst ofthe Present Invention

The antibody of the present invention has reaction specificity to theglycoglycerophospholipid inherent in Mycoplasma fementans. Accordingly,the glycoglycerophospholipid in a specimen can be immunologicallymeasured by using the antibody of the present invention. In thisprocedure, GGPL-I and GGPL -III can be measured by using the antibodywhich has reaction specificity to both of GGPL-I and GGPL-III. GGPL-IIIcan be selectively measured by using the antibody which has reactionspecificity to GGPL-III. When GGPL-III is measured, GGPL-III obtained asdescribed above can be used as a standard substance. Theglycoglycerophospholipid in a specimen includes a lipid fractionextracted from the specimen originating from a living body.

Those applicable to the immunological measurement include ordinaryimmunological methods based on the use of the antibody, such as ELISAmethods and immunostaining methods. For example, theglycoglycerophospholipid in a specimen can be measured by allowing aspecimen solution to contact with a solid phase including theanti-glycoglycerophospholipid antibody bound thereto so that theglycoglycerophospholipid contained in the specimen solution is bound tothe antibody, separating and removing non-adsorptive components from thesolid phase, subsequently allowing a glycoglycerophospholipidoriginating from Mycoplasma fementans labeled with a label substance tocontact with the solid phase, making a competitive reaction between theglycoglycerophospholipid contained in the specimen solution and thelabeled glycoglycerophospholipid, and detecting any one of the labelsubstance bound to the solid phase and the label substance not bound tothe solid phase.

Alternatively, the glycoglycerophospholipid in a specimen can bemeasured by allowing a specimen solution and a glycoglycerophospholipidlabeled with a standard substance to contact with a solid phaseincluding the anti-glycoglycerophospholipid antibody bound thereto,allowing the glycoglycerophospholipid contained in the specimen solutionand the labeled glycoglycerophospholipid to make a competitive reactionwith the antibody, and detecting any one of the label substance bound tothe solid phase and the label substance not bound to the solid phase. Inthis procedure, a non-labeled standard glycoglycerophospholipid may beused in place of the labeled glycoglycerophospholipid to perform acompetitive reaction between the glycoglycerophospholipid in thespecimen and the standard glycoglycerophospholipid, followed by allowingthe anti-glycoglycerophospholipid antibody labeled with a labelsubstance to contact with the solid phase so that any one of the labelsubstance bound to the solid phase and the label substance not bound tothe solid phase is detected. A labeled secondary antibody may be alsoused in this procedure.

Further, it is allowable that the glycoglycerophospholipid in a specimensolution is bound to a solid phase, with which a labeledanti-glycoglycerophospholipid antibody is allowed to contact so that anyone of the label substance bound to the solid phase and the labelsubstance not bound to the solid phase is detected.

It is also allowable that a standard glycoglycerophospholipid is boundto a solid phase, with which a specimen solution and a labeledanti-glycoglycerophospholipid antibody are allowed to contact so thatany one of the label substance bound to the solid phase and the labelsubstance not bound to the solid phase is detected. A labeled secondaryantibody may be also used in this procedure. GGPL-III in a specimen canbe measured by using purified GGPL-III as the standardglycoglycerophospholipid.

Further, the anti-glycoglycerophospholipid antibody in a specimen can bemeasured by allowing a specimen solution to contact with a solid phaseincluding the anti-glycoglycerophospholipid bound thereto so that theanti-glycoglycerophospholipid antibody contained in the specimensolution is bound to the antibody, separating and removingnon-adsorptive components from the solid phase, subsequently making areaction with a secondary antibody obtained by labeling an anti-humanimmunoglobulin antibody with a label substance, and detecting the labelsubstance. The glycoglycerophospholipid of the present invention isinherent in Mycoplasma fementans. Accordingly, it is possible to knowthe presence or absence of infection with Mycoplasma fementans byinspecting the presence or absence of the anti-glycoglycerophospholipidantibody in a specimen.

Besides the foregoing, various variations are known as methods for theimmunological measurement. Any of the methods can be applied to thepresent invention.

Other than the method based on the use of the solid phase as describedabove, those adoptable in the present invention include methods to beused for immunologically measuring haptens and antibodies, such as aliquid phase method comprising the steps of allowing aglycoglycerophospholipid in a specimen and a labeledglycoglycerophospholipid to make a competitive reaction with theantibody described above, separating the antigen bound to the antibodyfrom the free antigen by using, for example, polyethylene glycol,dextran, or a secondary antibody, and detecting a label substance of thefree labeled antigen.

Those usable as the solid phase include ordinary materials such asagarose beads, latex particles, and wells of microtiter plates composedof, for example, polystyrene or nylon, regardless of their forms (forexample, particles, fine particles, test tubes, microtiter plates, andstrips). It is preferred to perform blocking by using, for example, BSA(bovine serum albumin) or gelatin after the antibody or theglycoglycerophospholipid is bound to the solid phase. Those usable asthe label substance include, for example, enzymes capable of colordevelopment of a dye based on an enzyme reaction, such as peroxidase andalkaline phosphatase; radioisotopes; and fluorescent dyes such asfluorescein isothiocyanate.

As for the dye, for example, 4-chloro-1-naphthol, O-phenylenediamine(OPD), or 3,3′-diaminobenzidine is used for peroxidase, andp-nitrophenylphosphate is used for alkaline phosphatase.

Mycoplasma fementans has the glycoglycerophospholipid according to thepresent invention. Therefore, Mycoplasma fementans can be detected byusing the anti-glycoglycerophospholipid antibody of the presentinvention. For example, Mycoplasma fementans in a specimen can bedetected by extracting a lipid fraction from the specimen, allowing theextracted lipid fraction to contact with a solid phase so that the lipidis adsorbed to the solid phase, reacting the solid phase including thelipid adsorbed thereto with the antibody of the present invention,simultaneously or subsequently making a reaction with ananti-immunoglobulin antibody labeled with a label substance and reactivewith the antibody, and detecting the label substance.

Further, the glycoglycerophospholipid concerning the present inventionis inherent in Mycoplasma fermentans. Therefore, Mycoplasma fementanscan be also detected by measuring a glycoglycerophospholipid containedin a specimen in accordance with the method as described above, andrelating the presence or absence of the glycoglycerophospholipid or anexisting amount thereof to the presence or absence of Mycoplasmafermentans or an existing amount thereof in the specimen.

Those usable as the specimen in the method for detecting theglycoglycerophospholipid or the method for detecting Myconlasmafermentans described above include, for example, blood, serum, plasma,cerebro-spinal fluid, urine, synovial fluid, and cultured cell solution(supernatant).

Besides the foregoing methods, Mycoplasma fermentans can be alsodetected by reacting the anti-glycoglycerophospholipid antibody labeledwith a label substance, with a tissue or cells of a living organismexactly or after applying a treatment for immobilizing aglycoglycerophospholipid, binding the labeled antibody to the tissue orcells of the living organism infected with Mycoplasma fementans, anddetecting the label substance. Those usable as a method for theimmobilizing treatment include, for example, methods based on the use offormalin, glutaraldehyde, and paraformaldehyde. Alternatively,Mycoplasma fementans may be also detected by reacting a non-labeledanti-glycoglycerophospholipid antibody instead of theanti-glycoglycerophospholipid antibody labeled with the label substancewith a tissue or cells of a living organism subjected to an immobilizingtreatment, simultaneously or subsequently making a reaction with asecondary antibody obtained by labeling an antibody againstimmunoglobulin of an immunized animal with a label substance, theantibody against the immunoglobulin of the immunized animal having beenprepared by using an animal other than the immunized animal used toprepare the antibody, binding the labeled secondary antibody to thetissue or cells of the living organism infected with Mycoplasmafementans, and detecting the label substance.

Upon detection of the glycoglycerophospholipid or Mycoplasma fementanscontained in a specimen by immunological methods, the detection can beconveniently performed by previously preparing a reagent kit comprisingthe antibody having reaction specificity to the glycoglycerophospholipidof Mycoplasma fementans, and a secondary antibody obtained by labelingan antibody against immunoglobulin of an immunized animal with a labelsubstance, the antibody against the immunoglobulin of the immunizedanimal being prepared by using an animal other than the immunized animalused to prepare the antibody having reaction specificity to theglycoglycerophospholipid of Mycoplasma fementans.

The kit is specifically exemplified by a kit comprising, for example, amicrotiter plate, a blocking reagent such as BSA (bovine serum albumin),the glycoglycerophospholipid of Mycoplasma fementans (standardsubstance), the antibody of the present invention, a peroxidase-labeledanti-mouse IgG antibody, an aqueous solution of hydrogen peroxide, OPD,and a washing buffer. It is preferred that the antibodies, theglycoglycerophospholipid of Mycoplasma fementans, and other pertinentcomponents of the kit are provided as lyophilized preparations ordissolved solutions in a solvent capable of stably storing them.

The detection of Mycoplasma fementans can be utilized for the followingdiagnosis methods.

(1) Prediction of Crisis of Retrovirus Infectious Disease

It is reported that Myconlasma fermentans is an exacerbation factor ofAIDS (acquired immunodeficiency syndrome) (Lo, S. -C. et al., 1993, Res.Microbiol., 144, 489-493). Crisis of AIDS does not necessarily occureven when a patient is infected with HIV (human immunodeficiency virus).Usually, AIDS has a long latent period, and complex infection ofMycoplasma fermentans participates in crisis of AIDS. It is conceivedthat Mycoplasma fementans also participates in crisis of infectiousdiseases caused by other retroviruses.

Lipids extracted from various species of mycoplasmas were added to aculture liquid of cells latently infected with HIV, at certain lipidconcentrations (final concentrations) as shown in Table 1, and the cellswere cultured. A number of destroyed cells was inspected by using anoptical microscope, which was used as an index of frequency of inductionof expression of HIV. Results are shown in Table 1.

TABLE 1 Species of Lipid concentration Inducing Mycoplasma (μg/ml)activity M. fermentans 550 +++ M. hyorhinis 310 ± M. orale 1090  − M.salivarium 1090  − M. hominis 490 + M. arthritidis 600 −

According to the results and the foregoing report, it is understood thatMycoplasma fementans has an activity to induce proliferation of theretrovirus. Therefore, it is possible to predict the crisis byinspecting the presence or absence of complex infection of Mycoplasmafementans with, for example, blood of a patient infected with theretrovirus. Thus it is possible to take an appropriate treatment.

(2) DiaQnosis of Rheumatism

It is reported that Mycoplasma fementans is a cause of rheumatism(Williams, M. H. et al., Lancet ii: 277-280 (1970)). Therefore, it isexpected that morbidity of rheumatism can be diagnosed by detectingMycoplasma fementans by using the antibody of the present invention.

(3) Application to Targeting Therapy

It is expected to apply and utilize the antibody of the presentinvention for preventing crisis of retrovirus diseases and curingrheumatism such that an anti-HIV agent such as azidothymidine ordideoxyinosine and/or an anti-mycoplasmal agent is bound to the antibodyof the present invention to be administered to a patient infected withthe retrovirus or a patient of rheumatism.

(4) Utilization an Neutralizing Antibody for Treatment

The antibody of the present invention is expected to present an activityas a neutralizing antibody which binds to Mycoplasma fementans to avoidinfection caused by Mycoplasma fementans. Administration of the antibodyto a living body makes it possible to use the antibody for curing orpreventing infectious diseases caused by viruses having high possibilityof complex infection of Mycoplasma fementans.

(5) Diagnosis of Nephritis

As demonstrated in Examples described later on, a lipid, to which theanti-glycoglycerophospholipid antibody of the present invention binds,is highly frequently found in blood of patients of nephritis. It hasbeen suggested that nephritis can be diagnosed by detecting the lipid.The lipid is distinguished from GGPL-III judging from the position onHPTLC. However, the lipid is considered to be a lipid havingantigenicity similar to that of GGPL-III because the monoclonal antibodywhich recognizes GGPL-III binds to the lipid. The substance havingantigenicity similar to that of GGPL-III can be immunologically measuredby using the anti-glycoglycerophospholipid antibody of the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a result of electrophoresis of amplified products obtainedby first step PCR for spacer regions between 16S-23S ribosome RNA genesof various mycoplasmas, wherein:

1: Mycoplasma fementans GGPL strain separated from MT-4 cells;

2: Mycoplasma fementans PG18;

3: Myconlasma hyorhinis DBS1050;

4: Mycoplasma arginini G230;

5: Mycoplasma orale CH19299;

6: Mycoplasma salivarium PG20;

7: Mycoplasma penetrans GTU-54-6A1;

8: negative control.

FIG. 2 shows a result of electrophoresis of fragments obtained bydigesting, with various restriction enzymes, amplified products obtainedby second step PCR for a spacer region between 16S-23S ribosome RNAgenes of Mycoplasma fementans, wherein 1: VspI, 2: HindIII, 3: ClaI, 4:HincII, 5: HaeIII, 6: no treatment.

FIG. 3 shows a TLC pattern (densitometry) of phospholipids contained ina lipid fraction of Mycoplasma fementans.

FIG. 4 shows a TLC pattern of phospholipids and glycolipids contained inlipid fractions of MT-4 cells infected or not infected with Mycoplasmafementans, wherein PC indicates phosphatidylcholine, and SPM indicatessphingomyelin.

FIG. 5 shows an infrared absorption spectrum of GGPL-III.

FIG. 6 shows a liquid secondary ion mass spectrum of GGPL-III obtainedby the (+) method.

FIG. 7 shows a liquid secondary ion mass spectrum of GGPL-III obtainedby the (−) method.

FIG. 8 shows a tandem mass spectrum of GGPL-III obtained by the (+)method.

FIG. 9 shows a tandem mass spectrum of GGPL-III obtained by the (−)method.

FIG. 10 shows a unidimensional ¹H NMR spectrum.

FIG. 11 shows unidimensional ¹H NMR spectrums of 3-aminopropane-1,2-diol(1-aminopropane-2,3-diol) (A) and 2-aminopropane-1,3-diol (B).

FIG. 12 shows a two-dimensional ¹H NMR spectrum of GGPL-III obtained bythe PH-DQF-COSY method.

FIG. 13 shows a two-dimensional ¹H-³¹P NMR spectrum of GGPL-III.

FIG. 14 shows a result of TLC of phospholipids contained in lipidfractions of various mycoplasmas (stained with Dittmer reagent),wherein:

1: Mycoplasma fementans GGPL strain;

2: Mycoplasma fementans incognitus;

3: Mycoplasma fementans PG18 strain;

4: Mycoplasma fementans F17 strain;

5: Mycoplasma fementans F1 strain;

6: Mycoplasma fementans F7 strain;

7: MVcoplasma arthritidis;

8: Mycoplasma hominis.

FIG. 15 shows a result obtained by immunostaining a lipid fraction ofMT-4 cells separated by HPTLC, by using a polyclonal antibody describedin Example 2.

FIG. 16 shows a result obtained by immunostaining lipid fractions ofvarious mycoplasmas separated by HPTLC, by using a monoclonal antibodydescribed in Example 3 (reference numerals indicate the same contents asthose depicted in FIG. 14).

FIG. 17 shows a photograph illustrating a result obtained byimmunostaining MT-4 cells infected with Mycoplasma fementans, by usingthe monoclonal antibody of the present invention.

FIG. 18 shows results obtained by immunostaining lipids extracted fromblood samples of patients of nephritis or normal individuals andseparated by HPTLC, by using the monoclonal antibody described inExample 3, wherein “nephritis patients” indicates results for lipidsextracted from blood samples of patients of nephritis, and “normalindividuals” indicates results for lipids extracted from blood samplesof normal individuals.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be explained more specifically below inaccordance with Examples.

EXAMPLE 1 Isolation and Structural Analysis of NovelGlycoglycerophospholipid

<1> Cultivation of Mycoplasma fementans and Preparation of Lipids

A culture supernatant of MT-4 cells (human helper T cell strain infectedwith human T lymphotropic retrovirus Type I (HTLV-I)) containing GGPLswas filtrated with a filter having a pore size of 0.22 μm. An obtainedfiltrate was inoculated to an agar medium of PPLO broth (produced byDifco Laboratories) to perform cultivation. Colonies were isolated andthen cultivated in a liquid medium of PPLO broth (produced by DifcoLaboratories) containing 10% (v/v) fetal bovine serum (FBS), 5% (w/v)yeast extract (produced by Flow Laboratories), 1,000 units/mlpenicillin, 1% (w/v) dextrose, and 0.002% (w/v) phenol red. Formedcolonies were fried egg-shaped. Thus it was confirmed that the obtainedmicroorganism was a mycoplasma.

DNA was prepared from this microorganism. The spacer region between16S-23S ribosome RNA genes, which might differ depending on themycoplasmal species, was analyzed by means of two-step PCR (Harasawa, R.et al., 1993, Res. Microbiol., 144: 489-493) for respective mycoplasmalspecies of Mycoplasma fementans isolated from MT-4 cells, Mycoplasmafementans type strain PG18, Mycoplasma hyorhinis DBS1050 (ATCC 17981),Mycoplasma arginini G230 (ATCC 23838), Mycoplasma orale CH19299,Mycoplasma salivarium PG20 (ATCC 23064), and Mycoplasma penetransGTU-54-6A1. As a result, a product of first step PCR of thismicroorganism was coincident with that of PG18 strain as the type strainof Mycoplasma fermentans (FIG. 1). Further, an amplified product, whichwas obtained by second step PCR by using the PCR product, was digestedwith VspI and HindIII, and was not digested with ClaI, HincII, andHaeIII (FIG. 2). Thus this microorganism was identified to be Mycoplasmafermentans, which was designated as GGPL strain (M. fermentans GGPLstrain).

Methanol (100 ml) was added to microbial cells (1 g) of Mycoplasmafementans obtained by cultivation in accordance with the methoddescribed above, followed by being left to stand for several hours.Chloroform (200 ml) was added thereto, and the preparation was subjectedto an ultrasonic treatment, followed by being left to stand for severalhours. The preparation was homogenized with a Potter type Teflonhomogenizer to obtain a homogenate which was centrifuged at 10,000 rpmto recover a supernatant thereof. The supernatant was evaporated, andthus a lipid extract (50 mg) was obtained.

<2> Analysis of Lipid Fraction of Mycoplasma fementans

The lipid fraction of Mycoplasma fementans obtained as described abovewas applied to an HPTLC (high-performance thin layer chromatography)plate (produced by Merck), and developed with a mixed solvent ofchloroform: methanol: 0.2% (w/v) calcium chloride aqueoussolution=50:45:10 (v/v/v). The phospholipid was stained with Dittmerreagent. A phospholipid pattern was measured on the basis of absorptionat 580 nm by using a TLC densitometer (CS910, produced by Shimadzu). Asa result, six bands (Lipid i, Lipid ii, Lipid iii, Lipid iv, Lipid v,and Lipid vi) were detected (FIG. 3).

Lipid fractions were extracted from MT-4 cells infected with Mycoplasmafementans and MT-4 cells obtained by treating the former cells with ananti-mycoplasmal agent (MC201, produced by Dainippon Pharmaceutical) inaccordance with a known method (Bligh and Dyer, Can. J. Biochem.Physiol., 37, 911-917 (1959)). Respective extracts of the lipidfractions were applied to an HPTLC (high-performance thin layerchromatography) plate (produced by Merck), and developed with a mixedsolvent of chloroform: methanol: 0.2% (w/v) calcium chloride aqueoussolution =50:45:10 (v/v/v). The glycolipid was stained with the orcinolreagent, and the phospholipid was stained with Dittmer reagent. As aresult, two bands were detected, which were found in MT-4 (GGPL+), andwere not found in MT-4 (GGPL−) (FIG. 4). One of the two bands wasidentified to be GGPL-I with its structure having been already known,and the other was identified to be GGPL-III. Other Dittmerreagent-positive bands were identified to be GM2, GM1a, and GD1a, andother orcinol reagent-positive bands were identified to bephosphatidylcholine and sphingomyelin (Matsuda, K. et al., Biochem.Biophys. Acta, 1168, 123-129 (1993)).

A lipid fraction was extracted from MT-4 cells (treated with theanti-mycoplasmal agent PC201) cultured with addition of a culturesupernatant of MT-4 cells (infected with Mycoplasma fementans ) passedthrough a filter having a pore size of 0.22 μm. The extracted lipidfraction was analyzed by HPTLC in the same manner as described above. Asa result, the two bands described above were detected. Therefore, it isclear that the two bands, i.e., GGPL-I and GGPL-III originate fromMycoplasma fementans.

According to the behavior on TLC, it was revealed that GGPL-I andGGPL-III corresponded to Lipid v and Lipid vi described aboveoriginating from Mycoplasma fermentans respectively. According toresults of FAB mass spectrometry, it was confirmed that GGPL-I wasidentical with Lipid v, and GGPL-III was identical with Lipid vi.

Extraction was performed from MT-4 cells (60 ml, wet volume) infectedwith Mycoplasma fementans by using solvents of chloroform: methanol=2:1,1:1, and 1:2 (400 ml) to obtain a total lipid of 993 mg. The total lipidwas applied to a DEAE Sephadex A-25 column to separate it into anon-adsorptive fraction (neutral fraction) and an adsorptive fraction(acid fraction). Thus 775 mg of the non-adsorptive fraction wasobtained. The non-adsorptive fraction was applied to an Iatrobeadscolumn (produced by Iatron), and fractionated three times with aconcentration gradient of chloroform/methanol/water (83:16:0.5 to20:80:8, v/v/v). Finally, elution was performed with a concentrationgradient of 1-propanol/aqueous ammonia/water (80:5:15 to 75:5:20, v/v/v)to isolate 3 mg of GGPL-III.

<3> Structural Analysis of GGPL-III

GGPL-III was positive to the orcinol reagent, Dittmer reagent, andDragendorff reagent, and it was degraded by a treatment with mildalkali. GGPL-I was negative to the ninhydrin reaction, however, GGPL-IIIwas positive to the ninhydrin reaction. According to these results, itwas revealed that GGPL-III was a glycophospholipid containing choline.

Further, structural analysis of GGPL-III was performed as describedbelow.

(1) Measurement of Infrared Absorption Spectrum

An infrared absorption spectrum of GGPL-III was measured by using aninfrared spectrophotometer (FTIR -8100M, produced by Shimadzu) equippedwith an infrared microscope (IMS-8000, produced by Shimadzu). A resultis shown in FIG. 5.

As a result, absorption bands were detected, corresponding to —CH₂ groupand —CH₃ group (2957, 2920, 2852, 1467, 1419, 1378 cm⁻¹), hydroxyl group(3271 cm⁻¹), estercarbonyl group (1740, 1165 cm⁻¹), phosphate group(1091 cm⁻¹), choline group (970 cm⁻¹), and primary amine group (1560 to1670 cm⁻¹) respectively.

(2) Liquid secondary ion mass sPectrometry (LSIMS) GGPL-III purified asdescribed above (about 1 μg) was dissolved in a mixed solution (1 μL) ofchloroform: methanol (1 volume: 1 volume). 3-Nitrobenzyl alcohol in thecase of the (+) method or triethanolamine in the case of the (−) methodwas added and mixed to the solution, as a matrix in an amount of 0.5 mL.The obtained mixed solution was used as a sample to perform liquidsecondary ion mass spectrometric analysis by using a TSQ 70 triplequadrupole type mass spectrometer (produced by Finnegan MAT). Cesium ion(Cs⁺) accelerated to 20 keV was used as a primary ion flow. The spectrumwas obtained at a velocity of 250 atomic mass unit (amu)/sec. A spectrumobtained by the (+) method is shown in FIG. 6, and a spectrum obtainedby the (−) method is shown in FIG. 7.

As a result, ions were observed in the (+) method at m/z=1021, m/z=1049,and m/z=1077. According to this fact, it was suggested that at leastthree species of GGPL-III molecules having different fatty acidcompositions existed. It was concluded that a major component ofGGPL-III was represented by a peak of m/z=1049, having its molecularweight of 1048 obtained by subtracting a mass of proton of 1 from 1049.It was also concluded that the other components had molecular weights of1020 and 1076.

In the (−) method, ions were observed at m/z=1047, m/z=1076, andm/z=1103. Judging from combination with the spectrum analysis based onthe (+) method, it was suggested that at least four species of GGPL-IIImolecules having different fatty acid compositions existed. It wasconcluded that a major component of GGPL-III observed by the (−) methodwas represented by a peak of m/z=1047, having its molecular weight of1048 obtained by adding a mass of proton of 1 to 1047. It was alsoconcluded that another component had a molecular weight of 1104. Amolecular weight deduced from the ion of m/z=1076 was 1077. However, amolecular weight of 1076 was deduced from the result of the (+) method.A difference between this molecular weight and the molecular weight ofanother component corresponded to an amount of two methylene groups.Accordingly, it was concluded that the molecular weight was 1076.

(3) Tandem Mass Spectrometry (MS/MS)

Samples, which were prepared in the same manner as the samples used forLSIMS (a sample for the (+) method and a sample for the (−) method),were used to measure tandem mass spectrums by using a TSQ 70 triplequadrupole type mass spectrometer (produced by Finnegan MAT). Cesium ion(Cs⁺) accelerated to 20 keV was used as a primary ion flow. Algon, whichwas maintained at 0.26 pascal (2.0 mTorr), was used as a CAD (low energycollisionally activated dissociation) gas. The spectrum was obtained ata velocity of 250 atomic mass unit (amu)/sec. A spectrum obtained by the(+) method is shown in FIG. 8, and a spectrum obtained by the (−) methodis shown in FIG. 9.

As a result, ions were observed at m/z=184 and m/z=1049 in the (+)method. According to the presence of the ion of m/z=184, it wassuggested that phosphocholine was present in the molecule of GGPL-III.It was concluded that the molecular weight of GGPL-III was 1048 obtainedby subtracting a mass of proton of 1 from 1049. In the (−) method, anion of m/z=1047 was observed. According to this fact, it was concludedthat the molecular weight of GGPL-III was 1048 obtained by adding a massof proton of 1 to 1047. It was supported that among the pluralityspecies of GGPL-IIIs suggested by LSIMS, the molecule having themolecular weight of 1048 was the major component.

(4) Unidimensional ¹H NMR Spectrum

GGPL-III (500 μg) substituted with deuterium (²H), phosphatidylcholine(2 mg), and D-glucose 6-phosphate disodium salt (produced by OrientalYeast) (about 200 μg) were dissolved in 0.5 mL of a solvent composed of[²H] dimethyl sulfoxide ((C²H₃)₂SO): ²H₂O (98 volumes: 2 volumes)respectively to obtain ¹H-NMR spectrums at 60° C. at 400 MHz by using aGX-400 spectrometer (produced by JEOL). Tetramethylsilane was used as astandard.

An integrated value of a signal of glucose (GlcH1) was regarded as 1.00to standardize the spectrum, and intensities of the other signals arequantified. A result is shown in FIG. 10.

As a result, signals of glycerol (Gro) were detected at 4.319 ppm(GroH1b), 4.145 ppm (GroH1a), 5.118 ppm (GroH2), \3.677 ppm (GroH3b),and 3.562 ppm (GroH3a). Signals of choline were detected at 4.074 ppm(—POCH₂—), 3.529 ppm (—CH₂≡), and 3.139 ppm (—N(CH₃)₃). The spectrum ofGGPL-III was compared with unidimensional ¹H NMR spectrums obtained bythe foregoing method by using standard samples of3-aminopropane-1,2-diol (1-aminopropane-2,3-diol) and2-aminopropane-1,3-diol respectively (see FIGS. 11A and 11Brespectively). As a result, it was found that 2-aminopropane-1,3-diolwas highly possibly contained in GGPL-III. Further, a signal at 4.647ppm (GlcH1) was conspicuously detected as a signal of glucose (Glc).

(5) Two-dimensional ¹H NMR Spectrum

Samples prepared in the same manner as those used for the unidimensional¹H NMR spectrum were used to perform two-dimensional ¹H NMR analysis at60 ° C. at 400 MHz in accordance with the PH-DQF-COSY method with awidth of 2,000 Hz for each dimension by using an FX-400 spectrometer(produced by JEOL). A result is shown in FIG. 12.

As a result, signals of protons originating from diacylglycerol wereobserved (GroH2/H1b, GroH2/H1a, GroH2/H3b, GroH2/H3a in FIG. 12).Signals of protons of choline were observed (—CH₂—N—/—CH₂OP— in FIG.12).

(6) Two-dimensional ¹H-³¹ P NMR Spectrum

Samples prepared in the same manner as those used for the unidimensional¹H NMR were used to obtain a two-dimensional ¹H-³¹ P HMQC spectrum at60° C. at 400 MHz by using an FX-400 spectrometer (produced by JEOL). Aresult is shown in FIG. 13.

As a result, a spectrum equivalent to that obtained by ¹H NMR describedabove was obtained for the first dimension (¹H NMR), and two signalswere obtained from the second dimension (³¹ p NMR). This fact suggeststhat two phosphorus atoms (P) are contained in one molecule of GGPL-III.

No crossing peak was detected between positions in the first dimensionat which signals of 2-, 3-, 4-, and 5-protons of glucose residueappeared (in the vicinity of 2.9 to 3.0 ppm) and positions in the seconddimension at which signals of phosphorus appeared (−0.9 ppm and 1.1ppm). Accordingly, it was postulated that a compound containingphosphorus bound to 1-position or 6-position of glucose residue.Further, crossing peaks were detected between a signal of 6a-proton ofglucose residue (in the vicinity of 4.0 ppm in the first dimension) anda signal of phosphorus in the second dimension (−0.9 ppm), and between asignal of 6b-proton of glucose residue (in the vicinity of 3.6 ppm inthe first dimension) and signals of phosphorus in the second dimension(−0.9 ppm and 1.1 ppm) respectively.

Further, a crossing peak was detected between a signal of choline in thefirst dimension (about 4.05 to about 4.1 ppm) and a signal of phosphorusin the second dimension (−0.9 ppm). Accordingly, it was possible toassume a structure of phosphocholine in which phosphate group bound tocholine. Further, the crossing peak of phosphocholine deviated from thesignal of 6a-proton of glucose residue (in the vicinity of 4.0 ppm inthe first dimension). Accordingly, a structure was postulated, in whichphosphoric ester of aminopropanediol firstly bound to 6-position ofglucose residue, and phosphocholine further bound to phosphoric ester ofaminopropanediol.

Judging from summarization of the foregoing results, GGPL-III is a novelglycoglycerophospholipid having the following properties:

(A) the glycoglycerophospholipid is reactive with orcinol reagent,Dittmer reagent, Dragendorff reagent, and ninhydrin reagent;

(B) the glycoglycerophospholipid is degradable with alkali;

(C) the glycoglycerophospholipid is obtained as a non-adsorptivefraction upon fractionation with an anion exchanger having DEAE group;and

(D) the glycoglycerophospholipid has a molecular weight of 1048+28nmeasured by using a mass spectrometer, wherein n is −1, 0, 1, or 2.

Further, it has been demonstrated from the foregoing results thatGGPL-III comprises constitutional components ofα-glucopyranosyl-(1′-3)-1,2-diacyl-sn -glycerol, phosphocholine, andphosphoric ester of aminopropanediol. The phosphoric ester ofaminopropanediol is a phosphoric ester of 2-aminopropane-1,3-diol. It ispostulated that its binding site is 6′-position of glucose residue ofα-glucopyranosyl-(1′-3)-1,2-diacyl-sn-glycerol. Further, it is suggestedthat phosphocholine binds to the phosphoric ester of2-aminopropane-1,3-diol.

The respective GGPL-III molecules having the different molecular weightshave different types of acyl groups inα-glucopyranosyl-(1′-3)-1,2-diacyl-sn -glycerol. It is postulated thatany acyl group in the major component (molecular weight 1048) ispalmitoyl group. An entire deduced structure is one as represented bythe foregoing formula (I). The GGPL-III molecules having other molecularweights are different in length of the acyl group. It is postulated thatthe molecule having the molecular weight of 1020 has a myristoyl groupand a palmitoyl group, the molecule having the molecular weight of 1076has a palmitoyl group and a stearoyl group, and the molecule having themolecular weight of 1104 has two stearoyl groups or a palmitoyl groupand an eicosanoyl group. However, details are not clarified.

<4> Analysis of Lipid Components of Various Species of Mycoplasmas

Various species of mycoplasmas (Mycoplasma fermentans GGPL strain,Mycoplasma fementans incognitus (ATCC 53949), Mycoplasma fementans PG18strain, Mycoplasma fementans F17 strain, Mycoplasma fementans F1 strain,Mycoplasma fementans F7 strain, Mycoplasma arthritidis, and Mycoplasmahominis (ATCC 15488)) were cultivated in a liquid medium of PPLO broth(produced by Difco Laboratories) containing 10% (v/v) FBS (fetal bovineserum), 5% (w/v) yeast extract (produced by Flow Laboratories), 1,000units/ml penicillin, 1% (w/v) dextrose, and 0.002% (w/v) phenol red.Lipids were extracted from obtained culture liquids in accordance with aknown method (Bligh and Dyer, Can. J. Biochem. Physiol., 37, 911-917(1959)). The lipid extracts were applied to an HPTLC plate (produced byMerck), and developed with a mixed solvent of chloroform: methanol: 0.2%(w/v) calcium chloride aqueous solution=50:45:10 (v/v/v). The plate wasimmersed in 0.4% polyisobutylmethacrylic acid salt dissolved in hexanefor 30 seconds. The plate was stained with Dittmer reagent in accordancewith an ordinary method. A result is shown in FIG. 14. According to thisresult, the bands corresponding to GGPL-I and GGPL-III were found onlyin the respective species of Mycoplasma fementans, and the bands werenot found in other mycoplasmal species. According to this fact, it hasbeen concluded that GGPL-I and GGPL-III are glycoglycerophospholipidscharacteristic of Mycoplasma fementans.

EXAMPLE 2 Preparation of Anti-Mycoplasmal Glycolipid Polyclonal Antibody

Monophosphate lipid A (50 μg, produced by Ribi ImmunoChem Research) andcomplete adjuvant (50 μg, produced by nacalai tesque) were added to thelipid extract (50 mg) of Mycoplasma fementans obtained as describedabove, to which mineral oil (250 μl) was further added, followed bygrinding at 400 rpm for 2 minutes. Further, PBS containing 0.1% (v/v)Tween 80 (250 μl) was added thereto, followed by grinding at 400 rpm for2 minutes to obtain an emulsion (0.5 ml) containing the lipid extract.

The emulsion prepared by the foregoing method was subcutaneouslyinjected to 7-weeks-old female BALB/c mice in an amount of 0.5 ml perone individual. Two weeks and three weeks after the primingimmunization, the emulsion containing the lipid extract prepared by theforegoing method was intraperitoneally injected in an amount of 0.5 mlper one individual.

A serum was separated from blood of the mice immunized as describedabove, in accordance with an ordinary method. The serum was used toperform immunostaining as follows. A lipid fraction was extracted fromGGPL-positive MT-4 cells in accordance with a known method (Bligh andDyer, Can. J. Biochem. Physiol., 37, 911-917 (1959)). The extract of thelipid fraction was applied to a high-performance thin layerchromatography (HPTLC) plate (produced by Merck), and developed with amixed solvent of chloroform: methanol: 0.2% (w/v) calcium chlorideaqueous solution=50:45:10 (v/v/v). The plate was immersed in 0.4%polyisobutylmethacrylic acid salt dissolved in hexane for 30 seconds,and then dried in air.

The foregoing serum or a serum of a non-immunized mouse was added as aprimary antibody to the HPTLC plate, followed by incubation at 4° C.overnight. The HPTLC plate was washed with PBS. After that, aperoxidase-labeled anti-rabbit IgG antibody (produced by Cappel) wasadded as a secondary antibody to the HPTLC plate, followed by incubationat room temperature for 4 hours. The HPTLC plate was washed with purewater. After that, bands of antigens were detected by using a peroxidasecolor development kit (KONICA Immunostaining HRPKit, produced byKonica). A result is shown in FIG. 15. According to this result, it wasconfirmed that the foregoing serum contained a polyclonal antibodyagainst GGPL-I and GGPL-III.

EXAMPLE 3 Preparation of Anti-GGPL-III Monoclonal Antibody

(1) Preparation of Hybridoma

An emulsion containing the lipid extract of Mycoplasma fementans wassubcutaneously injected to 7-weeks-old female BALB/c mice in an amountof 0.5 ml per one individual in the same manner as described in Example2. Two weeks and three weeks after the priming immunization, theemulsion containing the lipid extract prepared by the foregoing methodwas intraperitoneally injected in an amount of 0.5 ml per oneindividual.

Four days after the final immunization, spleens were excised from themice, and a cell-floating suspension was prepared by using RPMI 1640medium. The spleen cells (2×10⁸ cells) were mixed with mouse myelomaSP2/0 cells at the logarithmic growth phase (azaguanine-resistant,IgG-non-secretable; ATCC CRL -1581, 2×10⁷ cells), followed bycentrifugation to obtain a residual precipitate to which 45%polyethylene glycol (PEG-4000, produced by Wako Pure Chemical, 1 ml) wasadded over 1 minute with mild shaking, followed by incubation for 2minutes at 37° C. with mild shaking. RPMI 1640 medium (1 ml) was addedthereto over 1 minute, followed by mild shaking. The same medium (1 ml)was added thereto over 1 minute, followed by mild shaking. After that,the same medium (8 ml) was further added thereto over 3 minutes.

After centrifuging the mixed cell suspension, the cells were floated inRPMI 1640 medium (50 ml) containing 10% fetal calf serum (FCS). Thesuspension was dispensed and poured into wells of four 96-wellmicroplates in an amount of 100 μl per one well. The cells were culturedin a carbon dioxide gas incubator (5% carbon dioxide gas, 37° C.). After24 hours, the medium was exchanged to HAT medium (10% (v/v) FCS mediumcontaining hypoxanthine, aminopterin, and thymidine), and the cells werecontinuously cultured in a carbon dioxide gas incubator (5% carbondioxide gas, 37° C.). Four days after the start of cultivation in theHAT medium, fresh HAT medium was added in an amount of 100 μl per onewell. Seven days after the start of cultivation in the HAT medium, themedium was exchanged to HT medium (prepared by removing aminopterin fromHAT medium). On the day following the exchange, the medium was exchangedto RPMI 1640 medium containing 10% (v/v) FCS, and then the presence orabsence of colony formation was checked.

(2) Selection of Antibody-producing Hybridoma

The lipid fraction of Mycoplasma fementans (20 μg) was dissolved in 10ml of ethanol to prepare a solution which was added to wells of 96-wellmicroplate in an amount of 50 μl per one well, followed by being driedfor 30 minutes with a dryer. PBS containing 1% (w/v) bovine serumalbumin (BSA) was added thereto in an amount of 100 μl per one well,followed by being left to stand at room temperature for 1 hour. Thewells were washed five times with a solution of 0.3 M sucrose in anamount of 100 μl per one well. After that, culture supernatants obtainedfrom the cultures described above were added to the respective wells inan amount of 100 μl per one well, followed by shaking at roomtemperature for 60 minutes. The respective wells were washed with asolution of 0.05% Tween 20. After that, a peroxidase-labeled anti-mouseIgG antibody (produced by Cappel) diluted 500 times with PBS was addedto the wells in an amount of 50 μl per one well, followed by shaking atroom temperature for 60 minutes.

The respective wells were washed with a solution of 0.05% Tween 20.After that, a citrate buffer containing 10 mg of ortho-phenylenediamineand 50 μl of 30% (v/v) hydrogen peroxide aqueous solution per 10 ml ofthe buffer was added to the wells in an amount of 100 μl per one well,followed by incubation at room temperature for 15 minutes. Sulfuric acid(0.5 M) was added to the wells in an amount of 100 μl per one well tostop the enzyme reaction of peroxidase, and then the absorbance at 490nm was measured by using a microplate reader.

Antibody-producing hybridomas were subjected to repeated cloning inaccordance with the limiting dilution method. Primary screening wasperformed by means of ELISA based on the use of the antigen of the lipidfraction of Mycoplasma fementans. Colonies positive in ELISA werefurther subjected to secondary screening by means of immunostaining forthe lipid fraction of Mycoplasma fementans on HPTLC. Thus a hybridomastrain, i.e., MF-III-1 strain was obtained, which produced a monoclonalantibody having reaction specificity to GGPL-III. This strain wasdeposited in National Institute of Bioscience and Human Technology ofAgency of Industrial Science and Technology of Ministry of InternationalTrade and Industry under a deposition number of FERM P-14324,transferred to international deposition based on the Budapest Treaty onMay 26, 1995, and awarded a deposition number of FERM BP-5115.

(3) Preparation of Monoclonal Antibody

The monoclonal antibody against GGPL-III was obtained by culturing theMF-III-1 strain in RPMI 1640 medium containing 10% (v/v) FCS, andrecovering its culture supernatant.

EXAMPLE 4 Evaluation of Monoclonal Antibody by Immunostaining

Immunostaining was performed as described below by using the obtainedanti-GGPL-III monoclonal antibody. Lipid fractions of various species ofmycoplasmas (Mycoplasma fementans GGPL strain, Mycoplasma fermentansincognitus (ATCC 53949), Mycoplasma fermentans PG18 strain, Mycoplasmafementans F17 strain, Mycoplasma fementans F1 strain, Mycoplasmafermentans F7 strain, Mycoplasma arthritidis, and Mycoplasma hominis(ATCC 15488)) were extracted in accordance with a known method (Blighand Dyer, Can. J. Biochem. Physiol., 37, 911-917 (1959)). The extractsof the lipid fractions were applied to a high-performance thin layerchromatography (HPTLC) plate (produced by Merck), and developed with amixed solvent of chloroform: methanol: 0.2% (w/v) calcium chlorideaqueous solution=50:45:10 (v/v/v).

The plate was immersed in 0.4% (w/v) polyisobutylmethacrylic acid saltdissolved in hexane for 30 seconds, and then dried in air. The anti-GGPL-III monoclonal antibody described above was added as a primary antibodyto the HPTLC plate, followed by incubation at 4° C. overnight. The HPTLCplate was washed with PBS. After that, a peroxidase-labeled anti-mouseIgG antibody (produced by Cappel) was added as a secondary antibody tothe HPTLC plate, followed by incubation at room temperature for 4 hours.The HPTLC plate was washed with pure water. After that, bands of theantigen was detected by using a peroxidase color development kit (KONICAImmunostaining HRPKit, produced by Konica). A result is shown in FIG.16.

According to this result, it is clear that the monoclonal antibody ofthe present invention specifically binds to the glycoglycerophospholipidof Mycoplasma fementans, and does not bind to phospholipids andglycolipids of Mycoplasma arthritidis and Mycoplasma hominis. As for thebands stained on HPTLC with Dittmer reagent and the orcinol reagent, theband of GGPL-I and the bands identified to be GM2, GM1a, GD1a,phosphatidylcholine, and sphingomyelin were not found in theimmunostaining. Thus the monoclonal antibody of the present inventionspecifically bound to only GGPL-III.

Next, MT-4 cells infected with Mycoplasma fermentans were immunostainedwith the anti-GGPL-III monoclonal antibody as a primary antibody, and afluorescein isothiocyanate (FITC)-labeled anti-mouse IgG antibody as asecondary antibody, followed by observation with a fluorescentmicroscope. A result is shown in FIG. 17.

According to this result, it is clear that MycoPlasma fermentans can bedetected by using the monoclonal antibody of the present invention.

EXAMPLE 5 Detection of GGPL-III or Substance Having Similar Antigenicityto GGPL-III in Blood of Nephritis Patient

Serums were prepared from blood samples of nephritis patients and normalindividuals in accordance with an ordinary method. Total lipids in theserums were extracted from the serums (each 100 μl) with a mixed solventof chloroform: methanol (2:1, 1:1, and 1:2 (V/V), each 100 μl) inaccordance with a known method (Bligh and Dyer, Can. J. Biochem.Physiol., 37, 911-917 (1959)). The extracting solvent was divided intothree layers, of which the lowermost layer was recovered. The recoveredfraction was dialyzed against water, and then lyophilized. Samplesobtained as described above were applied to a high-performance thinlayer chromatography (HPTLC) plate (produced by Merck), and developedwith a mixed solvent of chloroform: methanol: 0.2% (W/V) calciumchloride aqueous solution (50:45:10 (V/V/V)). Purified GGPL-III was usedas a standard substance, which was applied and developed on the HPTLCplate in the same manner as described above.

The plate was immersed in 0.4% (w/v) polyisobutylmethacrylic acid saltdissolved in hexane for 30 seconds, and then dried in air. The anti-GGPL-III monoclonal antibody described above was added as a primary antibodyto the HPTLC plate, followed by incubation at 4° C. overnight. The HPTLCplate was washed with PBS. After that, a peroxidase-labeled anti-mouseIgG antibody (produced by Cappel) was added as a secondary antibody tothe HPTLC plate, followed by incubation at room temperature for 4 hours.The HPTLC plate was washed with pure water. After that, bands of theantigen was detected by using a peroxidase color development kit (KONICAImmunostaining HRPKit, produced by Konica).

The test was performed in accordance with the method as described abovefor 9 specimens of serums of nephritis patients and 9 specimens ofserums of normal individuals. As a result, a signal was observed in thevicinity of the position of GGPL-III for 6 specimens of the 9 specimensof the nephritis patients (FIG. 18). This signal was not detected forthe specimens of the normal individuals (FIG. 18). The signal, which wasfrequently found in nephritis patients, was different from GGPL-IIIjudging from the position of HPTLC. However, it is assumed that thesignal represents a lipid having similar antigenicity.

Alternatively, after lipids are extracted as described above, thefollowing steps may be adopted. Namely, the lipids are lyophilized toprepare samples. Each of the samples is dissolved in a mixed solvent ofchloroform: methanol: water (83:16:0.5 (V/V/V)), and applied toIatrobeads column (equilibrated with a mixed solvent of chloroform:methanol: water (83:16:0.5 (V/V/V)) charged with Iatrobeads 6RS-8060(produced by Iatron). Stepwise elution is performed by changing thecomposition of chloroform: methanol: water to obtain an eluted fractionto be applied to HPTLC.

EXAMPLE 6 Detection of Anti-GGPL-III Antibody in Serum by ELISA Methodby Using Purified GGPL-III as Antigen

GGPL-III (40 μg) purified in Example 1 was dissolved in 10 ml of ethanolto prepare a solution which was added to wells of 96-well microplate inan amount of 50 μl per one well, followed by being dried with a dryerfor 30 minutes. PBS containing 1% (w/v) bovine serum albumin (BSA) wasadded in an amount of 100 μl per one well, followed by beingstationarily left to stand at room temperature for 1 hour. The plate waswashed five times with a solution of 0.3 M sucrose in an amount of 100μl per one well. After that, serums prepared in accordance with anordinary method from blood samples of normal individuals, AIDS patients,nephritis patients, and HTLV-I associated myelopathy (HAM) patientsrespectively were added to the wells in an amount of 100 μl per onewell, followed by shaking at room temperature for 60 minutes. Therespective wells were washed with a solution of 0.05% Tween 20. Afterthat, a peroxidase-labeled anti-human IgG antibody (produced by Cappel)diluted 2,000 times with PBS was added to the wells in an amount of 50μl per one well, followed by shaking at room temperature for 60 minutes.

The respective wells were washed with a solution of 0.05% Tween 20.After that, a citrate buffer containing 10 mg of ortho-phenylenediamineand 50 μl of 30% (v/v) hydrogen peroxide aqueous solution per 10 ml ofthe buffer was added to the wells in an amount of 100 μl per one well,followed by incubation at room temperature for 15 minutes. Sulfuric acid(0.5 M) was added to the wells in an amount of 100 μl per one well tostop the enzyme reaction of peroxidase, and then the absorbance at 450nm was measured by using a microplate reader.

The absorbance at 450 nm was measured in accordance with the methoddescribed above for serums of normal individuals (46 specimens), AIDSpatients (10 specimens), nephritis patients (22 specimens), and HTLV-Iassociated myelopathy (HAM) patients (9 specimens). An absorbanceobtained from those of the normal individuals was used as a control.Serum specimens having absorbances at 450 nm larger than the normalindividuals were regarded as anti-GGPL-III antibody positive. A resultis shown in Table 2.

TABLE 2 Positive ratio for Name of disease anti-GGPL-III antibody Normalindividual 1/46  (2.2%) AIDS 8/10 (80.0%) Nephritis 3/22 (13.8%) HAM2/9  (22.2%)

As understood from Table 2, those which were anti-GGPL-IIIantibody-positive in serum were 1 specimen of the 46 specimens (2.2%)for the normal individuals, 8 specimens of the 10 specimens (80.0%) forthe AIDS patients, 3 specimens of the 22 specimens (13.8%) for thenephritis patients, and 2 specimens of the 9 specimens (22.2%) for theHTLV-I associated myelopathy patients respectively. Further,investigation was made by using a peroxidase-labeled anti-human IgMantibody (produced by Cappel) instead of the secondary antibodydescribed above (peroxidase-labeled anti-human IgG antibody). As aresult, the positive ratio for the anti-GGPL-III antibody in serums ofnephritis patients was further increased.

INDUSTRIAL APPLICABILITY

According to the present invention, the novel glycoglycerophospholipidoriginating from Mycoplasma fermentans, i.e., GGPL-III is obtained.Further, the antibody having reaction specificity to theglycoglycerophospholipid originating from Mycoplasma fermentans,especially the polyclonal antibody having reaction specificity to GGPL-Iand GGPL-III, and the monoclonal antibody having reaction specificity toonly GGPL-III are obtained.

The use of the antibody of the present invention makes it possible tospecifically detect Mycoplasma fermentans. It is expected to apply theantibody of the present invention to prediction of crisis of retrovirusinfectious diseases such as AIDS, diagnosis of rheumatism, and diagnosisof nephritis.

Further, the antibody of the present invention has a possibility to beused for curing or preventing infectious diseases caused by viruseswhich highly possibly undergo complex infection of Mycoplasmafermentans.

What is claimed is:
 1. An isolated anti-glycoglycerophospholipidantibody immunolooically reactive with a glycoglycerophospholipidcomprising at least phosphocholine, glucose, fatty acid, and glycerol,the lipid being non-adsorptive to an anion exchanger havingdiethylaminoethyl group, and unstable against alkali, wherein theglycoalycero-phospholipid is a glycoglycerophospholipid specificallyexisting in Mycoplasma fermentans.
 2. The anti-glycoglycerophospholipidantibody according to claim 1, which immunoloically reacts with both of6′-O-phosphocholine-α-glucopyranosyl-(1′-3)-1,2-diacyl-sn-glycerol and aglycoglycerophospholipid purified from Mycoplasma fermentans, having thefollowing properties: (A) the glycozlycerophospholipid is reactive withorcinol reagent, Dittmer reagent, Dragendorff reagent and ninhydrinreagent; (B) the glycoglycerophospholipid is degradable with alkali; (C)the glycozylcerophospholipid is obtained as a non-adsorptive fractionupon fractionation with an anion exchanger having diethylaminoethylgroup; and (D) the glycozylycerophospholipid has a molecular weight of1048+28n measured by using a mass spectrometer, wherein n is −1, 0, 1 or2.
 3. The anti-glycoglycerophospholipid antibody according claim 1,which is a polyclonal antibody.
 4. The anti-glycoglycerophospholipidantibody according to claim 1, which immunologically reacts with only aglycoglycerophospholipid purified from Mycoplasma fermentatans, havingthe following properties: (A) the glycoglycerophospholipid is reactivewith orcinol reagent, Dittmer reagent, Dragendorff reagent, andninhydrin reagent; (B) the glycoglycerophospholipid is deoradable withalkali; (C) the glycoglycerophospholipid is obtained as a non-adsorptivefraction upon fractionation with an anion exchanger havingdiethylaminoethyl group; and (D) the glycoglycerophospholipid has amolecular weight of 1048+28n measured by using a mass spectrometer,wherein n is −1, 0, 1, or
 2. 5. The anti-glycoglycerophospholipidantibody according to claim 1, which is a monoclonal antibody or afragment thereof.
 6. The anti-glycoglycerophospholipid antibodyaccording to claim 1, which specifically recognizes Mycoplasmafementans, and does not recognize other species of mycoplasmas.
 7. Theanti-glycoglycerophospholipid antibody according to claim 6, wherein theother species of mycoplasmas are Mycoplasma arthritidis and Mycoplasmahominis.
 8. The anti-glycoglycerophospholipid antibody according toclaim 1, which makes no cross reaction with sialic acid-containingglycolipid (ganglioside), platelet-activating factor(1-alkyl-2-acetylglycero-3-phosphocholine) or a partially deacylatedproduct thereof, phosphatidylcholine or a partially deacylated productthereof, and sphingomyelin.
 9. A method for measuring aglycoglycerophospholipid, comprising the step of immunologicallymeasuring the glycoglycerophospholipid having the following propertiescontained in a specimen, by using the anti-glycoglycerophospholipidantibody as defined in claim 1: the glycoglycerophospholipid comprisesat least phosphocholine, glucose, fatty acid, and glycerol, the lipidbeing non-adsorptive to an anion exchanger having diethylaminoethylgroup, and unstable against alkali.
 10. A method for measuring asubstance, which is immunologically reactive with the isolatedanti-glycoglycerophospholipid antibody of claim 1, contained in aspecimen, comprising the step of immunologically measuring the substanceby measuring the binding to the anti-glycoglycerophospholipid antibody.11. A method for measuring a glycoglycerophospholipid contained in aspecimen, comprising the steps of allowing a specimen solution tocontact with a solid phase including the anti-glycoglycerophospholipidantibody as defined in claim 1 bound thereto so that theglycoglycerophospholipid contained in the specimen solution is bound tothe antibody, separating and removing non-adsorptive components from thesolid phase, subsequently allowing a glycoglycerophospholipidoriginating from Mycoplasma fementans labeled with a label substance tocontact with the solid phase, making a competitive reaction between theglycoglycerophospholipid contained in the specimen solution and thelabeled glycoglycerophospholipid, and detecting any one of the labelsubstance bound to the solid phase and the label substance not bound tothe solid phase.
 12. A method for detecting Mycoplasma fementanscontained in a specimen, comprising the steps of extracting a lipidfraction from the specimen, allowing the extracted lipid fraction tocontact with a solid phase so that the lipid is adsorbed to the solidphase, reacting the solid phase including the lipid adsorbed theretowith the anti-glycoglycerophospholipid antibody as defined claim 1,simultaneously or subsequently making a reaction with a secondaryantibody obtained by labeling an antibody against immunoglobulin of animmunized animal with a label substance, the antibody against theimmunoglobulin of the immunized animal having been prepared by using ananimal other than the immunized animal used to prepare theanti-glycoglycerophospholipid antibody, and detecting the labelsubstance.
 13. A method for detecting Mycoplasma fementans, comprisingthe steps of reacting the anti-glycoglycerophospholipid antibody asdefined in claim 1 labeled with a label substance, with a tissue orcells of a living organism exactly or after applying a treatment forimmobilizing a glycoglycerophospholipid, binding the labeled antibody tothe tissue or cells of the living organism infected with Mycoplasmafementans, and detecting the label substance.
 14. A method for detectingMycoplasma fementans, comprising the steps of reacting theanti-glycoglycerophospholipid antibody as defined in claim 1 with atissue or cells of a living organism exactly or after applying atreatment for immobilizing a glycoglycerophospholipid, simultaneously orsubsequently making a reaction with a secondary antibody obtained bylabeling an antibody against immunoglobulin of an immunized animal witha label substance, the antibody against the immunoglobulin of theimmunized animal having been prepared by using an animal other than theimmunized animal used to prepare the anti-glycoglycerophospholipidantibody, binding the labeled secondary antibody to the tissue or cellsof the living organism infected with Mycoplasma fementans, and detectingthe label substance.
 15. A reagent kit for detecting Mycoplasmafermentans or a glycoglycerophospholipid of Mycoplasma fermentanscontained in a specimen in accordance with an immunological method,comprising the anti-glycoglycerophospholipid antibody as defined inclaim 1, and a glycoglycerophospholipid of Mycoplasma fementans labeledwith a label substance.
 16. A reagent kit for detecting Mycoplasmafermentans or a glycoglycerophospholipid of Mycoplasma fermentanscontained in a specimen in accordance with an immunological method,comprising the anti-glycoglycerophospholipid antibody as defined inclaim 1, and a secondary antibody obtained by labeling an antibodyagainst immunoglobulin of an immunized animal with a label substance,the antibody against the immunoglobulin of the immunized animal beingprepared by using an animal other than the immunized animal used toprepare the anti-glycoglycerophospholipid antibody.
 17. A method formeasuring a glycoglycerophospholipid specifically existing in Mycoplasmafementans in a specimen, comprising the step of immunologicallymeasuring the glycoglycerophospholipid by using theanti-glycoglycerophospholipid antibody as defined in claim
 1. 18. Amethod for detecting Mycoplasma fementans, comprising the steps ofmeasuring a glycoglycerophospholipid contained in a specimen inaccordance with the measuring method as defined in claim 17, andrelating the presence or absence of the glycoglycerophospholipid or anexisting amount thereof to the presence or absence of Mycoplasmafementans or an existing amount thereof in the specimen.
 19. The methodfor detecting Mycoplasma fementans according to claim 18, wherein theglycoglycerophospholipid contained in the specimen is a lipid fractionextracted from the specimen originating from a living organism.
 20. Themethod for detecting Mycoplasma fementans according to claim 19, whereinthe specimen originating from the living organism is blood, serum,plasma, cerebro-spinal fluid, urine, synovial fluid, or cultured cellsolution.